Many biological materials that are prepared for human, veterinary, diagnostic and/or experimental use may contain unwanted and potentially dangerous biological contaminants or pathogens, such as viruses, bacteria, nanobacteria, yeasts, molds, mycoplasmas, ureaplasmas, prions and parasites. Consequently, it is of utmost importance that any biological contaminant or pathogen in the biological material be inactivated before the product is used. This is especially critical when the material is to be administered directly to a patient, for example in blood transfusions, blood factor replacement therapy, organ transplants and other forms of human therapy corrected or treated by intravenous, intramuscular or other forms of injection. This is also critical for the various biological materials that are prepared in media or via culture of cells or recombinant cells which contain various types of plasma and/or plasma derivatives or other biologic materials and which may be subject to mycoplasma, prion, bacterial and/or viral contaminant or pathogens.
Most procedures for producing biological materials have involved methods that screen or test the biological materials for one or more particular biological contaminants or pathogens rather than removal or inactivation of the contaminant(s) or pathogen(s) from the material. Materials that test positive for a biological contaminant or pathogen are merely not used. Examples of screening procedures include the testing for a particular virus in human blood from blood donors. Such procedures, however, are not always reliable and are not able to detect the presence of certain viruses, particularly in very low numbers. This reduces the value or certainty of the test in view of the consequences associated with a false negative result. False negative results can be life threatening in certain cases, for example in the case of Acquired Immune Deficiency Syndrome (AIDS). Furthermore, in some instances it can take weeks, if not months, to determine whether or not the material is contaminated. Therefore, it would be desirable to apply techniques that would kill or inactivate contaminants or pathogens during and/or after manufacturing the biological material.
Moreover, to date, there is no reliable test or assay for identifying prions within a biological material that is suitable for screening out potential donors or infected material. This serves to heighten the need for an effective means of destroying priors within a biological material, while still retaining the desired activity of that material.
In conducting experiments to determine the ability of technologies to inactivate viruses, the actual viruses of concern are seldom utilized. This is a result of safety concerns for the workers conducting the tests, and the difficulty and expense associated with the containment facilities and waste disposal. In their place, model viruses of the same family and class are used.
In general, it is acknowledged that the most difficult viruses to inactivate are those with an outer shell made up of proteins, and that among these, the most difficult to inactivate are those of the smallest size. This has been shown to be true for gamma irradiation and most other forms of radiation as these viruses' diminutive size is associated with a small genome. The magnitude of direct effects of radiation upon a molecule are directly proportional to the size of the molecule, that is the larger the target molecule, the greater the effect. As a corollary, it has been shown for gamma-irradiation that the smaller the viral genome, the higher the radiation dose required to inactive it.
Among the viruses of concern for both human and animal-derived biological materials, the smallest, and thus most difficult to inactivate, belong to the family of Parvoviruses and the slightly larger protein-coated Hepatitis virus. In humans, the Parvovirus B19, and Hepatitis A are the agents of concern. In porcine-derived materials, the smallest corresponding virus is Porcine Parvovirus. Since this virus is harmless to humans, it is frequently chosen as a model virus for the human B19 Parvovirus. The demonstration of inactivation of this model parvovirus is considered adequate proof that the method employed will kill human B19 virus and Hepatitis A, and by extension, that it will also kill the larger and less hardy viruses such as HIV, CMV, Hepatitis B and C and others.
More recent efforts have focussed on methods to remove or inactivate contaminants in the products. Such methods include heat treating, filtration and the addition of chemical inactivants or sensitizers to the product.
Heat treatment requires that the product be heated to approximately 60° C. for about 70 hours which can be damaging to sensitive products. In some instances, heat inactivation can actually destroy 50% or more of the biological activity of the product.
Filtration involves filtering the product in order to physically remove contaminants. Unfortunately, this method may also remove products that have a high molecular weight. Further, in certain cases, small viruses may not be removed by the filter.
The procedure of chemical sensitization involves the addition of noxious agents which bind to the DNA/RNA of the virus and which are activated either by UV or other radiation. This radiation produces reactive intermediates and/or free radicals which bind to the DNA/RNA of the virus, break the chemical bonds in the backbone of the DNA/RNA, and/or cross-link or complex it in such a way that the virus can no longer replicate. This procedure requires that unbound sensitizer is washed from products since the sensitizers are toxic, if not mutagenic or carcinogenic, and cannot be administered to a patient.
Irradiating a product with gamma radiation is another method of sterilizing a product. Gamma radiation is effective in destroying viruses and bacteria when given in high total doses (Keathly et al., “Is There Life After Irradiation? Part 2,” BioPharm July-August, 1993, and Leitman, “USe of Blood Cell Irradiation in the Prevention of Post Transfusion Graft-vs-Host Disease,” Transfusion Science 10:219-239 (1989)). The published literature in this area, however, teaches that gamma radiation can be damaging to radiation sensitive products, such as blood, blood products, protein and protein-containing products. In particular, it has been shown that high radiation doses are injurious to red cells, platelets and granulocytes (Leitman). U.S. Pat. No. 4,620,908 discloses that protein products must be frozen prior to irradiation in order to maintain the viability of the protein product. This patent concludes that “[i]f the gamma irradiation were applied while the protein material was at, for example, ambient temperature, the material would be also completely destroyed, that is the activity of the material would be rendered so low as to be virtually ineffective”. Unfortunately, many sensitive biological materials, such as monoclonal antibodies (Mab), may lose viability and activity if subjected to freezing for irradiation purposes and then thawing prior to administration to a patient.
Recently, public attention has been attracted to the problem of human and animal products containing biological contaminants or pathogens that cause transmissible spongiform encephalopathies (TSEs) in mammals. TSEs cause inflammation and characteristic spongelike holes in the delicate membranes surrounding brain cells, which results in loss of coordination, dementia, and, eventually, death. Perhaps the best-known TSE is bovine spongiform encephalopathy (BSE), more popularly known as mad cow disease. BSE made headlines in 1996 when about a million cattle in the United Kingdom became infected with the disease when they ate feed made from the processed animal parts of infected sheep, pigs, and chickens. Ingestion of the infected cow meat caused about 20 people in Britain to develop an unusual form of Creutzfeldt-Jakob disease. Other TSEs include kuru, a rare disease contracted by natives of New Guinea who ate the infected brains of their dead relatives during ritual cannibalism, and scrapie, which affects sheep and goats and is so named because diseased sheep sometimes scrape off their own wool.
A prion (a shortened term for proteinaceous infectious particle) is believed to be a small protein associated with TSEs in cows, sheep, humans, and other mammals. Prions appear to be a mutated form of a normal protein. The normal protein (PrP) is found on the surface of nerve cells in the brain, white blood cells, muscle cells, and cells of many other tissues. The role of the normal protein is not yet understood, but its structure has been elucidated. A hundred times smaller than the smallest virus, the normal protein is composed of 208 amino acids twisted into three α-helices, from one of which extends a floppy tail of 97 amino acids. The mutated form of the protein (PrPsc) is built of the same amino acids. Instead of α-helices, however, the mutated protein is folded into β-sheets.
The high level of the mutated protein in neural and other tissue of an infected individual makes transmission of infection to another individual more likely, particularly if the non-infected individual consumes the tissue(s) of the infected individual. In this manner, infection with TSEs has occurred in animals fed processed tissue(s) obtained from infected animals, in humans who consumed tissue(s) obtained from infected animals or humans, and in humans who received tissue(s) or tissue extracts therapeutically and the tissue(s) had been donated by an infected individual.
In 1967, Tikvah Alper and her colleagues at the Hammersmith Hospital in London extracted brain tissue from scrapie-infected sheep. This processed tissue was then injected into healthy sheep to see if the disease would be transmitted. The healthy sheep contracted scrapie, indicating that the infectious agent was in the diseased brain tissue and that it could reproduce in healthy animals to cause disease. Alper then exposed similar scrapie-infected tissue extracts to ultraviolet radiation, which normally destroys DNA and RNA, and found that the extracts maintained their ability to transmit scrapie. The resistance of the infectious agent to ultraviolet radiation suggested that neither a virus nor bacteria, both of which reproduce through nucleic acids, caused the disease.
In the early 1980s, Stanley B. Prusiner at the University of California, San Francisco concluded that proteins were responsible for TSEs based on evidence that tissue extracts from scrapie-infected animals no longer caused disease after exposure to treatments known to destroy proteins. It was suggested that the mutated protein causes disease when it contacts the normal protein and triggers part of it to switch from the normal α-helical form to the mutant β-pleated form. A chain reaction would follow, resulting in the cluster of tangled, nonfunctional plaques found in the brains of animals that die from TSEs.
Not all scientists and doctors agree, however, that TSEs are transmitted by an infectious protein. It is still considered possible that the infectious agent responsible for TSEs is a small virus, either alone or in combination with the prion protein or another, as yet unidentified, cofactor. For the purposes of the present invention, the actual nature of the agent(s) responsible for TSEs is unimportant, so long as the agent(s) are rendered inactive, i.e. non-infectious, by the processes of the present invention, while retaining an adequate level of the activity of the treated biological material.
There therefore remains a need for methods of sterilizing compositions of biological materials that are effective for reducing or preventing the occurrence of TSEs without an adverse effect on the biological material(s).